1. Technical Field
The present invention relates to a method and apparatus for performing determination of immune reactants in biological fluids and, more particularly, for performing in vitro semi-quantitative determinations of allergen-specific (e.g., IgE, IgG, etc.) antibodies in human serum.
2. Discussion of the Prior Art
It is known that, in humans, immediate type allergic reactions (e.g., hay fever, extrinsic asthma, or atopic eczema) are mediated by reaginic antibodies belonging to the IgE class of immunoglobulins. Atopic individuals exposed to allergens such as pollens, dust or animal danders, produce specific IgE antibodies against these allergens. The present invention is concerned with determining the presence of circulating allergen-specific IgE antibodies in the blood plasma or serum of an affected individual.
A technique for accomplishing this result is known in the prior art as described in U.S. Pat. No. 3,720,760 (Bennich et al), the disclosure of which is expressly incorporated herein, in its entirety, by this reference. Specifically, Bennich et al disclose an in vitro method for analyzing a test sample (e.g., a body fluid such as blood serum or blood plasma) by contacting, in vitro, the test sample with a water insoluble polymer to which a test allergen has been bound. A reaction takes place between the test allergen on the polymer and the reagin-IgE directed against the allergen so that the reagin is bound to the test allergen on the insoluble polymer. The polymer, in sheet form, and with the test allergen and the reagin-IgE attached thereto,may then be contacted with antibodies against reagin-IgE that have been labeled with a radiation-emitting atom or group. The insoluble polymer sheet is separated from the fluid, whereupon the radiation emitted from the insoluble polymer with the substances attached thereto, or the radiation emitted from the separated fluid, is measured. If the reagin-IgE directed against the allergen is present in the sample, labeled reagin is bound to the insoluble phase which then emits radiation. The latter increases with increasing concentration of the reagin-IgE in the test sample. The radiation of the liquid phase decreases with increasing concentration of the reagin-IgE as more labeled reagin is bound to the insoluble phase. The measured radiation values obtained in this procedure for the test sample can be compared with values for control samples. If instead of radioimmunoassay (RIA) techniques, one were to use enzyme-immunoassay (EIA), color intensity, rather than radiation, becomes the measured parameter.
The use of polymer sheets as a vehicle to which the test allergen is bound, and to which the allergen-specific antibodies attach, becomes unwieldy in practice. To overcome the problem, the invention disclosed in my U.S. Pat. No. 4,891,321 employs multiple individual test units, each in the form of an elongated rod having an allergen-coated tip at its distal end. The proximal end of each rod is engaged in a support strip so that the test units are disposed in a position-identified linear array. Spacing between the test units matches the spacing between reaction containers in an assembly of such containers. The test units are inserted into respective reaction containers containing test serum. If there is an antibody specific to the allergen coated on any of the tips, that allergen-specific antibody becomes bound to the associated allergen in the corresponding reaction container. The tips are then washed, dried without rubbing, and inserted into a second set of reaction containers in which a suitable enzyme-labelled antibody conjugate has been poured. The test units are removed and rinsed once again and then placed into a third set of reaction containers containing chromogenic substrate that develops a specific color upon positive reaction. After incubation, the test units are removed and the remaining liquid in each reaction container is analyzed for color development and intensity by a suitable spectrophotometric analyzer.
This technique is reliable and greatly simplifies the portion of the Bennich et al method during which the polymer, to which a test allergen has been bound, is inserted into the test serum to permit circulating allergen specific IgE antibodies to attach to the bound allergen. However, it is desirable to reduce the time required to perform the method described in my prior patent and to eliminate the need for a costly spectrophotometric analyzer.
Another test apparatus known in the prior art for providing in vitro determinations of immune reactants in biological fluids is marketed by Quidel of San Diego, California as the Allergy Screen. This apparatus includes a fibrous test strip having successive pads or sections impregnated with specific allergens. The test strip is inserted into a test tube containing a serum or whole blood sample and left to incubate at room temperature for six hours or more. The manufacturer describes a quick test procedure involving only a thirty minute incubation period, but the sensitivity of this test is not acceptable for most procedures. After incubation the test strip is washed and then inserted into a second test tube containing conjugate solution for thirty minutes. Thereafter the test strip is washed again and then inserted into a third test tube containing a substrate for an additional thirty minutes. Upon removal from the third test tube the test strip is blotted on a paper towel until all pads are dry. The test strip is then mounted on a test result card with each test pad disposed adjacent a corresponding test strip. If the blue color intensity of any individual allergen test pad is greater than the intensity of a negative control pad, a positive result has been obtained. If the blue color intensity of a test pad is equal to or lighter than the intensity of the negative control pad, a negative result has been obtained.
The above-described Quidel Allergy Screen procedure takes at least seven hours for meaningful results and is not as sensitive as desired in that it tends to miss borderline allergic conditions. The reason for these problems appears to relate to the use of a test strip made of fibrous material such as cellulose paper. It requires a relatively long time for the reactants to penetrate the fibrous material and, once penetration occurs, to be washed out. In fact, once the reactants or dye penetrates the material, it is very difficult to rinse them out. The result is that both specific and non-specific dye gets trapped in the test strip.